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Previous Article | Next Article 
Journal of Virology, February 2008, p. 1701-1713, Vol. 82, No. 4
0022-538X/08/$08.00+0 doi:10.1128/JVI.02157-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Marjorie B. Kovler Viral Oncology Laboratories, The University of Chicago, 910 East 58th Street, Chicago, Illinois 60637
Received 2 October 2007/ Accepted 20 November 2007
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ABSTRACT |
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UL41-infected cells gE is retained in the trans-Golgi network and is not widely dispersed in cellular membranes. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(i) In wild-type virus-infected cells, there is not significant colocalization of ICP0 and ICP4. ICP0 initially colocalizes with the ND10 structure (12, 30, 31), fills the nucleus, and eventually, between 9 and 12 h after infection, is present in a diffuse form entirely in the cytoplasm (16, 19, 20, 28). Export of ICP0 to the cytoplasm is blocked by exposure of cells 2 h after infection to proteasomal inhibitor MG132 (13, 28). A striking feature of the translocation process is that exposure of infected cells to MG132 after ICP0 has been exported to the cytoplasm results in the relocation of ICP0 to the nucleus (28).
ICP0 is also exported to the cytoplasm in cells infected with mutants lacking ICP4 (ICP4) (16, 28). Unlike the situation with wild-type virus-infected cells, ICP0 is translocated at earlier times after infection and forms small dense structures dispersed throughout the cytoplasm or arranged around the nucleus. In the presence of MG132, added late after infection, ICP0 is retained in the cytoplasm in similar small dense structures that also contain proteasomal components (28). Relevant to this report is the observation that a D199A substitution in ICP0 prevents the translocation of ICP0 to the cytoplasm (52, 53). This observation suggests that the translocation of ICP0 is an active process involving an interactive partner.
Last, ICP0 and ICP4 have been reported to colocalize in the cytoplasm in cells transfected with plasmids encoding the two proteins (35).
(ii) Although the focus of numerous publications has been on gE, this glycoprotein most likely exists in a complex with gI. A key property of this complex is that of an Fc receptor. gE consists of an 
396-residue ectodomain, a 25-residue transmembrane domain, and a 106-residue cytoplasmic domain (40). gI consists of a 248-residue ectodomain, a transmembrane domain, and a 94-residue cytoplasmic domain (47). gE and gI form a heterodimer. The gE domains required for association with gI and binding with immunoglobulin G (IgG) are in the ectodomain (2, 3, 4, 40). Both gE and gI cytoplasmic domains contain amino acid motifs associated with endocytosis, and both glycoproteins are highly phosphorylated by both viral and cellular kinases (1, 4, 34, 37, 54).
The phenotypic properties of the gE/gI complex have been studied extensively. gE/gI shuttles between the cell surface and the trans-Golgi network (4, 14, 32). A rich and persuasive literature has linked the gE/gI complex to cell-to-cell spread of virus. In experimental animals, the gE/gI complex plays a key role in neuronal spread across synapses (7, 8). The complex is not involved in virus entry or cell-cell fusion. Its action may well be to facilitate the delivery of virion cargo from the site of cytoplasmic envelopment to the cell surface (39). It has also been proposed that the binding of IgG by the Fc receptor diverts the immune system from cell destruction by antibody and complement (17, 29, 36, 51).
(iii) The UL41 protein, also known as the virion host shutoff protein, is an endoribonuclease that is active primarily early in infection upon entry of the virus into the cell. The UL41 protein of HSV-1 degrades RNA by endonucleolytic cleavage in the absence of other cellular or viral proteins (22, 46, 48, 50).
UL41 is expressed late in the replicative cycle. The newly synthesized UL41 protein is in complexes with VP16 and VP22 and is not active as a nuclease (21, 23, 45, 49). Numerous studies have linked components of the Fc receptor with members of the UL41/VP16/VP22 complex or other viral proteins. Specifically, the cytoplasmic domain of gE interacts with a conserved domain of VP22 (38). The interaction takes place in infected cells, since VP22 as well as UL11 was precipitated in connection with full-length gE/gI and also with gD from HSV-1-infected cells (15). HSV-1 VP22 mutants incorporate reduced amounts of gB, gD, and gE into the virion envelope (9, 11). It is noteworthy, however, that VP22 mutants lack functional UL41 protein (44). In essence, in the course of the replicative cycle, the gE/gI complex interacts with various tegument proteins that include VP22 and by extension the product of the UL41 gene.
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MATERIALS AND METHODS |
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HSV-1(F), a limited-passage isolate, is the prototype strain used in this laboratory (10). R7032 (gE-2), a recombinant virus with a HincII-NruI deletion in the coding sequence for the US8 and US8.5 genes, has been reported previously (33). R7030 (gE-1), a recombinant virus that contains an insertion of the chimeric 
27-TK gene into the unique HpaI site of the gE gene, has been described elsewhere (26). The construction and properties of the UL41 (VHS) mutant virus R2621 were reported elsewhere (41). The HSV-1 (KOS)d120 mutant, a kind gift of N. DeLuca, lacks both copies of the 4 gene and was grown in a Vero-derived cell line (E5) expressing the 4 gene. It also does not express the US3 gene (25).
Plasmid construction.
The plasmid pRB123, which carries a 6,584-bp BamHI J fragment (42), was digested to completion with the BstEII enzyme to delete domains of the US7 (gI) and US8 (gE) genes. The DNA was selfligated to generate the plasmid designated pRB123-BstEII collapsed. The 1,311-bp fragment from the pRB123/BstEII digest, which carries sequences from the US8 gene and only the carboxyl-terminal domain from the US7 protein, was ligated back to the pRB123-BstEII collapsed plasmid to generate the plasmid designated pgI, which carries a repaired US8 gene (as in pRB123) and a 531-bp deletion in the US7 (gI) coding sequence.
Construction of recombinant viruses.
Recombinant R7081 was constructed by cotransfection of rabbit skin cells with intact R7030 (gE-1) viral DNA and the pgI plasmid. TK– progeny viruses were selected on 143TK– cells overlaid with Dulbecco's modified Eagle medium containing 5% newborn calf serum and 40 µg of bromodeoxyuridine per ml of medium, as described previously (43). The recombinant viruses were plaque purified after two rounds of selection, and their purity was verified by PCR amplification of the entire gI coding sequence, which was expected to be 531 bp shorter than that of the wild-type gene. The restoration of gE protein expression and the abrogation of gI protein expression from the progeny virus were confirmed by immunoblot analysis, as detailed in Results.
Immunofluorescence analysis. The immunofluorescence studies were done as described earlier (18), with minor modifications. Briefly, cells grown on four-well slides were exposed for 2 h to 10 PFU of wild-type or mutant viruses per cell. For the experiments with MG132 (Biomol), the drug was added to the cultures at a final concentration of 5 µM after 2 h of absorption. Cells were fixed in 4% paraformaldehyde and, at the indicated time after infection, neutralized with 100 mM glycine in phosphate-buffered saline (PBS), permeabilized/blocked with 0.1% Triton X-100 in PBS in the presence of 20% horse serum and 1% bovine serum albumin (BSA) (PBS-TBH solution), and reacted with primary antibodies diluted in PBS-TBH, as described earlier (18). The ICP4 and ICP0 mouse monoclonal antibodies (Goodwin Institute for Cancer Research, Plantation, FL) and the ICP0 exon 2 rabbit polyclonal antibody (20) were used in a dilution of 1:2,000. The rabbit polyclonal antibody against the 20S proteasome core subunits (PW 8155; Affiniti Research Products Ltd., Mamhead, Exeter, Devon, United Kingdom) was used in a dilution of 1:3,000. The TGN46 rabbit polyclonal antibody (Abcam, Inc.) was used in a dilution of 1:500. The gE mouse monoclonal antibody (clone 1108; Goodwin Institute for Cancer Research, Plantation, FL) was used in a dilution of 1:500. Following this incubation, the samples were rinsed several times with PBS-TBH and reacted with the proper secondary antibody, Alexa Fluor 594-conjugated goat anti-rabbit or Alexa Fluor 488-conjugated goat anti-mouse, diluted 1:1,000 in PBS-TBH. Finally, after several rinses first with PBS-TBH and then with PBS, the samples were mounted in Vectashield mounting medium for fluorescence (Vector Laboratories) and examined with a Zeiss confocal microscope equipped with software provided by Zeiss.
Immunoblot analysis. Protein preparation and immunoblotting were done as detailed elsewhere (18). Briefly, replicate cell cultures in six-well plates were either mock infected or exposed to 10 PFU of virus per cell in 199V medium (Sigma) at 37°C. The cells were harvested at the indicated times after infection, rinsed with PBS, solubilized in triple detergent buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% Nonidet P-40, 0.5% sodium deoxycholate, 100 µg ml–1 of phenylmethylsulfonyl fluoride) supplemented with phosphatase inhibitors (10 mM NaF, 10 mM β-glycerophosphate, 0.1 mM sodium vanadate) and protease inhibitor cocktail (Sigma) as specified by the manufacturer, and briefly sonicated. The protein concentration in total cell lysates was determined with the aid of a Bio-Rad protein assay (Bio-Rad Laboratories), according to directions provided by the supplier. Approximately 70 µg of proteins per sample was subjected to further analysis.
Proteins were electrophoretically separated in denaturing polyacrylamide gels, electrically transferred to a nitrocellulose sheet, blocked with PBS supplemented with 0.02% (vol/vol) Tween 20 (PBST) and 5% nonfat milk, and reacted overnight at 4°C with the appropriate primary antibodies diluted in PBST-1% nonfat milk. The mouse monoclonal antibodies for ICP4, ICP0, and ICP8 (Goodwin Institute for Cancer Research, Plantation, FL) were used in a dilution of 1:1,000. The gI mouse monoclonal antibody (clone Fd69) (27) was used in a dilution of 1:250. The gE mouse monoclonal antibody (clone 3114), kindly provided by D. Johnson, was used in a dilution of 1:500. The anti-UL41 rabbit polyclonal antibody has been described before (50) and was used in a dilution of 1:1,000. The UL38 rabbit polyclonal antibody was purchased from the Goodwin Institute for Cancer Research and was used in a dilution of 1:1,000. The β-actin mouse monoclonal antibody (Sigma) was used in a dilution of 1:2,000. After several rinses with PBST-1% nonfat milk, the membranes were reacted with the appropriate secondary antibody conjugated to alkaline phosphatase. Finally, protein bands were visualized with 5-bromo-4-chloro-3-indolylphosphate (BCIP)-nitroblue tetrazolium (Denville Scientific, Inc.).
[35S]methionine labeling of infected cells. Replicate cultures in 25-cm2 flasks were either mock infected or exposed to 10 PFU of virus per cell and incubated at 37°C. At 7, 9, and 11 h after infection, the cells were rinsed with methionine-free, serum-free 199 medium (Sigma) and overlaid with 1 ml of this medium supplemented with 100 µCi of [35S]methionine (specific activity, >1,000 Ci/mmol; Perkin Elmer Life Sciences) for 1 h. The cells were harvested and solubilized, and approximately 100 µg of total proteins per sample was subjected to electrophoresis in an 8% denaturing polyacrylamide gel, transferred to a nitrocellulose sheet as described above, and subjected to autoradiography.
Electron microscopy.
HEp-2 cells were harvested at 18 h after infection with 10 PFU of either the HSV-1(F) or the gE-2 virus per cell, rinsed with serum-free culture medium, and fixed in 4% paraformaldehyde-0.15% glutaraldehyde in 0.1 M PB buffer (76.7 mM Na2HPO4, 26.4 mM NaH2PO4, pH 7.4) for 1 h. The fixed cells were rinsed three times for 20 min each with 0.1 M PB on ice and dehydrated in a graded ethanol series (30, 50, 70, 95, and 3x 100% ethanol in water) at 4°C (30 and 50%) and at room temperature (70, 95, and 3x 100%) for 15 min per step. Subsequently, the cells were infiltrated using a mixture of LR White medium (London Resin Company Limited) with 100% ethanol, 1:1, followed by infiltration with pure LR White medium overnight and five 1-h changes with pure LR White medium the next day. All infiltrations were done at room temperature. After infiltration, cells were polymerized in a vacuum oven at 45°C for 48 h. Ultrathin sections of the embedded cells, 80 nm, were made using a diamond knife (Reichert-Jung Ultra-cut E) and collected on Formvar-coated 200-mesh nickel grids.
For immunogold labeling, sections collected on grids were rehydrated with PBS for 30 min, blocked with 1% BSA in PBS for 30 min, and incubated with diluted ICP0 rabbit polyclonal antibody (e.g., 1:100, 1:500, 1:1,000, and 1:2,000 in 1% BSA in PBS) in a humidified chamber for 3.5 h at room temperature. Following incubation, the sections were washed six times for 10 min each with PBS, blocked again with 0.5% BSA in PBS for 25 min, and incubated with 10-nm colloidal gold-conjugated goat anti-rabbit IgG (heavy plus light chains; Ted Pella) diluted 1:10 in 0.5% BSA in PBS for 1 h in a humidified chamber at room temperature. Finally, the sections were washed with PBS three times for 10 min each, fixed with 1% glutaraldehyde in PBS for 10 min, aqua washed three times for 5 min each, stained briefly with uracyl acetate and lead citrate, air dried, and examined under a Siemens 102 electron microscope.
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RESULTS |
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gE, gI, and UL41 viruses.
In wild-type HSV-1-infected cells, ICP4 accumulates exclusively in the nucleus. In earlier studies, we noted that ICP4 accumulated in the cytoplasm of cells infected with gE or UL41 mutant virus (18). To further investigate this phenomenon, three series of experiments were done.
The objective of the first series of experiments centered on the role of gE and gI. In wild-type virus-infected cells, gE and gI form a complex, and it is generally thought that they function as a complex (2, 3, 7, 8). The question we addressed is whether ICP4 was translocated to the cytoplasm also in the absence of gI. For this purpose, we constructed a recombinant virus that lacks only the gI coding sequence. Figure 1 shows a schematic representation of procedures employed for this purpose. As the parent virus, we used gE-1 (R7030) (26). In this mutant, the coding sequence of gE was disrupted by the insertion into the unique HpaI site of the thymidine kinase open reading frame, driven by the 27 promoter (Fig. 1, line 6). In the next step, we repaired the gE coding sequence in gE-1 and simultaneously introduced a deletion of 531 bp in the gI coding sequence (Fig. 1, line 8). This recombinant was designated R7081.
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gE-1 contains an intact US8.5 gene. In most studies described in this report, we tested both recombinants and found them to give identical results.
In the second series of experiments, we examined the expression of gE and gI in cells infected with wild-type, gE, gI, or UL41 virus. The cells were harvested at 18 h after infection, solubilized, subjected to electrophoresis in denaturing gels, transferred to a membrane, and probed with antibody to gE or gI. The results were as follows.
In wild-type virus-infected cells, gE formed two fast-migrating, relatively sharp bands and a slow-migrating broad band (Fig. 2A, lane 2). In UL41 mutant virus-infected cells, the intensity of the two faster-migrating, sharp bands was decreased and the broad, slower-migrating band was more intense and migrated faster than that of the wild-type counterpart (Fig. 2A, lane 3). It would appear that gE is processed differently in the absence of UL41.
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gI mutant virus resembled to some extent that of gE in lysates of cells infected with the UL41 mutant virus. In brief, there was a decrease in the intensity of the two faster-migrating sharp bands. The diffuse, slow-migrating band became significantly broader and migrated faster (Fig. 2C, lane 5).
In wild-type virus-infected cells, gI forms two relatively sharp bands (Fig. 2B, lane 2). Both gE mutants yielded patterns of migration of gI very similar to those of gI in lysates of wild-type virus-infected cells (Fig. 2B, lanes 4 and 5, and C, lanes 3 to 4). gI present in lysates of UL41 mutant-infected cells formed a third, faster-migrating double band readily visible in lighter exposures (Fig. 2B, lane 3). It would appear that gI is also processed differently in the absence of UL41 than in the wild-type virus-infected cells.
Last, it was of interest to determine whether cells infected with mutant viruses differed with respect to the synthesis of viral proteins at approximately the time of translocation of ICP0 and ICP4 from the nucleus to the cytoplasm. In these experiments, HEL cells were mock infected or exposed to 10 PFU of wild-type, gE, gI, or UL41 virus per cell. At 7, 9, or 11 h after infection, the cells were rinsed extensively and labeled for 1 h with [35S]methionine, as detailed in Materials and Methods. Cells were then harvested, solubilized, transferred to a membrane, and subjected to autoradiography (Fig. 3A). After the autoradiographic images were collected, the membrane was cut and reacted with antibodies to ICP0, ICP8, UL38, gE, and UL41 (Fig. 3B). Examination of the autoradiographic images suggests that with minor exceptions the patterns of protein synthesis in cells infected with wild-type virus, gE, or gI at 7, 9, and 11 h after infection were very similar. In contrast, in cells infected with the UL41 mutant virus, the synthesis of proteins was much reduced and in the 11- to 12-h interval it was distinctly lower than in the preceding time intervals. These conclusions are borne out in the examination of total proteins accumulating in infected cells. Thus, we noted a reduction in the accumulation of ICP8 and UL38 protein (Fig. 3B).
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UL41, gE, or gI virus.
Replicate cultures of HEp-2 cells grown in four-well slides were exposed to 10 PFU of the indicated virus per cell. The cells were fixed at 2, 4, 6, 8, or 10 h after infection and stained with the ICP4 mouse monoclonal antibody, as detailed in Materials and Methods. The results from representative images shown in Fig. 4 and 5 were as follows. ICP4 was restricted to the nucleus in cells fixed at 2 and 4 h after infection. At later time intervals, the percentage of cells containing ICP4 in both the nucleus and the cytoplasm steadily increased. For example, in the gE mutant virus-infected cells, the percentage of cells exhibiting ICP4 in both the nucleus and the cytoplasm increased from 58.7% to over 95% between 6 and 10 h after infection. In UL41 mutant virus-infected cells, the percentage of cells exhibiting both nuclear and cytoplasmic accumulation of ICP4 increased from 60.5% to approximately 90%. In most of the cells, the accumulated cytoplasmic ICP4 formed dense structures (Fig. 4). In contrast, ICP4 was detected in the form of diffuse fluorescence in approximately 1.6% of wild-type virus-infected cells and the number increased to approximately 10% by 10 h. Figure 5 (right panel) summarizes the results obtained at 9 h after infection for the two gE mutants and the gI mutant virus. In essence, while in a small fraction of wild-type virus ICP4 was exported and could be visualized as a diffuse cytoplasmic fluorescence, in the vast majority of cells infected with the UL41, gE, or gI mutant virus ICP4 was exported and accumulated in the cytoplasm in the form of dense aggregates. We conclude that retention of ICP4 in the nucleus requires the presence in infected cells of functional gE, gI, and UL41 protein.
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UL41, gE, or gI mutant virus. In the first series of experiments, HEL cells, grown in four-well slides, were infected with wild-type or mutant viruses. The cells were fixed at 9 h after infection and reacted with anti-ICP0 antibody. The results shown in Fig. 5 (left panel) indicate that ICP0 was translocated to the cytoplasm in nearly 100% of cells infected with any one of the viruses tested. The difference between HSV-1(F) and mutant viruses centers on the appearance of ICP0. In wild-type virus-infected cells, ICP0 was diffused throughout the cytoplasm and ICP0 aggregates were found in only a very small fraction of cells. In contrast, in most cells infected with mutant viruses, ICP0 was aggregated and formed small dense structures. In cells fixed at 14 h after infection (data not shown), the aggregates were much larger, suggesting that the process continues for many hours.
The objective of the second series of experiments was based on the observation that both ICP0 and ICP4 were aggregated in the cytoplasm in mutant virus-infected cells. To test the hypothesis that the two proteins are coexported and coaggregated, HEL cells in four-well slides were infected with 10 PFU of wild-type, gE, or gI virus. The cells were fixed at 9 h after infection and reacted with monoclonal antibody to ICP4 and polyclonal antibody to ICP0. The results, shown in Fig. 6, were as follows. As expected, in HSV-1(F)-infected cells, ICP4 was retained in the nucleus whereas ICP0 was diffused throughout the cytoplasm (Fig. 6d to f). In both gE- and gI-infected cells, ICP4 was partially translocated to the cytoplasm. It appeared to be present in two forms, i.e., diffused throughout the cytoplasm and in the forms of small dense cytoplasmic structures (Fig. 6g and j). ICP0 was present in the cytoplasm, and it too appeared to be present in a diffuse form throughout the cytoplasm and in the form of small dense structures (Fig. 6h and k). In both instances, the aggregated structures of ICP0 colocalized with the structures of ICP4 (Fig. 6i and l).
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gE- or gI-infected cells both ICP0 and ICP4 are translocated and colocalize, at least partly, in dense structures.
The third series of experiments centered on the nature of the small dense nuclear structure containing ICP0. To resolve this question, HEp-2 cells were harvested 18 h after infection with wild-type or gE virus and processed for immunoelectron microscopy as described in Materials and Methods. The preparations were reacted with rabbit polyclonal antibody to ICP0 and anti-rabbit IgG antibody conjugated to gold. Representative images are shown in Fig. 7.
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(ii) In gE mutant virus-infected cells, these structures were also present in the cytoplasm. In every respect—size, distribution, grain density, and reactivity with the ICP0 antibody—the cytoplasmic structures could not be differentiated from the nuclear structures (Fig. 7C and D).
(iii) ICP0 antibody also reacted, albeit less strongly, with electron opaque material at the nuclear periphery, commonly described as marginated chromatin (Fig. 7E).
(iv) Last, it has been reported that virions contain ICP0 (55). In none of the many sections examined in this study did we observe gold particles in apposition to virions either in the vicinity of the nuclear membranes or in the cytoplasmic vesicles (Fig. 7E and F). It is conceivable that the amounts of ICP0 packaged in virions are too small to be detected by this method.
Properties of the cytoplasmic structures containing ICP0 and ICP4.
In wild-type virus-infected cells, ICP0 is retained in the nucleus in infected cells exposed to proteasomal inhibitors at early times (e.g., 2 h after infection). Conversely, exposure of infected cells after translocation (e.g., 9 h after infection) results in the translocation of cytoplasmic ICP0 back into the nucleus (28). Exposure of cells infected with the d120 mutant virus, lacking ICP4, at early times to proteasomal inhibitors results in nuclear retention of ICP0. Exposure to the inhibitors at late times results in aggregation of proteasomal components with ICP0 in the cytoplasm. Since in cells infected with gE, gI, or UL41 both ICP0 and ICP4 were translocated, the question arose as to whether the process of translocation and cytoplasmic accumulation in cells infected with these mutants is different from that taking place in cells infected with the ICP4 mutant virus. To resolve this question, HEL cells grown on four-well slide cultures were mock infected or exposed to 10 PFU of HSV-1(F), UL41, ICP4, gE, or gI mutant virus per cell. At 2 h after infection, a replicate set of cultures was treated with 5 µM MG132. The cells were fixed at 9 h after infection and reacted with mouse monoclonal antibody to ICP0 or rabbit polyclonal antibody to 20S proteasome core subunits. The results, shown in Fig. 8, were as follows.
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As previously reported, in cells infected with the ICP4 mutant virus, ICP0 was translocated to the cytoplasm and aggregated in small dense structures (28). In MG132-treated cells, ICP0 was retained in the nucleus (compare Fig. 8A, images 10 to 12, with B, images 10 to 12).
In cells infected with UL41, gI, or gE, as expected on the basis of data presented in this report, ICP0 was translocated to the cytoplasm and aggregated in small dense structures. In cells treated with MG132 at 2 h after infection, ICP0 was also translocated to the cytoplasm and aggregated in small dense cytoplasmic structures. In essence, in the cells infected with these mutants, MG132 failed to block the translocation of ICP0 (compare Fig. 8A, images 7 to 9, 13 to 15, and 16 to 18, with B, corresponding images).
As in the case of cells infected with the UL41, gI, or gE mutants and treated with proteasomal inhibitors, the antibody to the proteasomal core components reacted with the small cytoplasmic aggregates of ICP0. The antibody to the core components reacted with the surface of the aggregates and appeared to form a halo around these structures (Fig. 8). This halo effect was either too weak to be seen in or absent from untreated infected cells.
Subcellular distribution of glycoprotein E in UL41, gI, and gE mutant-infected cells.
Published reports have shown that early after infection the gE/gI complex is sorted to the trans-Golgi network, whereas late in infection the complex is redistributed from the trans-Golgi network to other sites (4, 14, 32). During these trafficking events, the localization of other proteins may also be affected, as is the case for gE-dependent translocation of the IP3 receptor I (18). To determine whether the subcellular localization of gE is affected in gI- or UL41-infected cells, HEL cells grown in four-well slides were mock infected or exposed to 10 PFU of HSV-1(F), gE, gI, or UL41 virus. The cells were fixed at 4, 8, or 12 h after infection and reacted with the anti-gE mouse monoclonal antibody and the anti-TGN46 rabbit polyclonal antibody. The results, shown in Fig. 9, were as follows. At 4 h after infection, gE was detected around the perinuclear area of all of the infected cells. The perinuclear gE protein colocalized with the trans-Golgi network marker TGN46. Newly synthesized protein was detected in the Golgi apparatus, on the nuclear membrane, and (diffuse) in the cytosol in all of the infected cells by 8 h after infection (data not shown). A clear difference emerged at 12 h after infection. While in most wild-type virus-infected cells gE was present in small aggregates in membranes or diffuse in the cytosol, in cells infected with either gI or UL41, gE was aggregated in the trans-Golgi network. This network was retained in both gI and UL41 mutants even late after infection. Our results indicate differences in localization of gE in cells infected with gI or UL41 mutant virus and delay in the corruption of the trans-Golgi network compared to results for the wild-type virus-infected cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(i) Foremost, ICP4 is a nuclear protein in wild-type virus-infected cells. Its function is required throughout the replicative cycle (5, 6). Translocation to the cytoplasm in the absence of gE, gI, or UL41 protein implies that its nuclear retention is due to an expressed function regulated by the virus and is not due solely to the nuclear localization signal embedded in the primary structure of the protein. It is noteworthy that Zhu et al. have noted that in Vero cells both ICP0 and ICP4 encoded by the KOS strain are also detected in the cytoplasm in the form of diffuse and punctuate fluorescence (57, 58). These investigators noted that ICP4 localizes in the nucleus in the absence of ICP27 or in cells infected with certain ICP27 mutants. In contrast, coexpression of ICP27 inhibited or reduced nuclear localization of ICP4 and ICP0. The inhibitory function was mapped to the carboxyl-terminal half of ICP27 (57, 58). In more recent studies, Lengyel et al. noted that leptomycin B causes massive export of ICP4 and ICP0 from the nucleus to the cytoplasm (24). They also reported that resistance to leptomycin B maps to residues 21 to 63 in the amino-terminal domain of ICP27 (24). Interestingly, the drug has no such effect in cells infected with leptomycin B-resistant mutants. Moreover, mutants resistant to leptomycin B, including the mutants lacking residues 21 to 63 of ICP27, cannot be differentiated from wild-type viruses with respect to the ICP27 shuttling between the nucleus and cytoplasm or viral replication. The appearances of accumulated cytoplasmic ICP0 and ICP4 are different, and no suggestion of colocalization of ICP0 and ICP4 is evident from the published results. The authors suggest that the function of the amino-terminal domain of ICP27 is to export ICP0 and ICP4.
(ii) At very early stages in viral replication, ICP0 is in the nucleus, first in association with ND10 nuclear structures and later dispersed though the nucleus (12, 30, 31). ICP0 is translocated to and dispersed throughout the cytoplasm at later stages of the replicative cycle as a consequence of its dynamic association with proteasomes, inasmuch as the proteasome inhibitor MG132 blocks the translocation of ICP0 to the cytoplasm or, in cells exposed to the inhibitor later in infection, it causes the protein to be translocated back to the nucleus (16, 20). The dynamic interaction of ICP0 with proteasomal components is also apparent from the observation that in cells infected with a mutant lacking ICP4, ICP0 is translocated to the cytoplasm earlier than in cells infected with wild-type mutants (28). In contrast to ICP0 of wild-type virus-infected cells, ICP0 of mutant virus-infected cells forms dense cytoplasmic structures that, in the presence of MG132, also contain proteasomal components.
The involvement of a viral protein in the translocation process may be inferred from the observation that inhibition of DNA synthesis significantly delays but does not abolish the translocation of ICP0 (28). This observation suggests that the function is expressed by a late protein (
1), whose synthesis is retarded but not abolished by inhibitors of DNA synthesis. The involvement of host or viral proteins in the translocation process may also be inferred from the observation that the D199A substitution in ICP0 precludes the export of ICP0 to the cytoplasm (52, 53).
The conclusions reached by Lengyel et al. (24) and Lopez et al. (28) may in fact not be contradictory. The cotransfection studies carried out by Zhu and Schaffer suggest that ICP27 blocks the import of rather then exports ICP0 or ICP4 (58). If the studies of Lengyel et al. (24) are interpreted in the same light, it could be argued that ICP27 monitors and blocks excessive accumulation of ICP0 and ICP4 in the nucleus by sequestering the nuclear import machinery for its own use. In contrast, the translocation of ICP0 late in infection in wild-type virus-infected cells and the translocation of ICP4 and ICP0 in cells infected with the gE, gI, or UL41 mutant are late events and involve physical translocation of protein from the nucleus to the cytoplasm and colocalization of ICP0, ICP4, and proteasomal components. The difference between the translocation of ICP0 in wild-type virus-infected cells and that taking place in mutant virus-infected cells is that neither ICP0 nor ICP4 is reimported into the nuclei of mutant virus-infected cells following exposure to proteasomal inhibitors. The implication of this finding is that the mechanisms of translocation may be different in the two systems.
(iii) Physical interactions between ICP0 and ICP4 have been reported previously (56). This report, however, is the first report of colocalization of the proteins outside the nucleus in the context of a productive infection. It is of interest that Mullen et al. (35) reported that ICP4 and ICP0 colocalized in the cytoplasm of cells transfected with plasmids encoding wild-type ICP4 and a mutant form of ICP0 lacking residues 365 to 517 which by itself localized in the cytoplasm. The authors concluded that ICP0 dominated the interaction with ICP4 and suppressed its nuclear localization signals. Our studies involve wild-type proteins in the context of productively infected cells, with the result that the two proteins were both exported to the cytoplasm and colocalized in dense structures. Since in wild-type virus-infected cells ICP0 is translocated independently of ICP4, the question arises whether in the absence of retention signals for ICP4, ICP0 acts as a chaperone for translocation and ultimately colocalization in the dense cytoplasmic bodies.
(iv) Examination of mutant and wild-type virus-infected cells by electron microscopy with gold particle-conjugated antibody revealed that the structures containing ICP0 and by extension ICP4 were dense, amorphous protein aggregates that were not enclosed by a membrane. Of particular interest was the observation that structures virtually identical in appearance were present in the nuclei but not in the cytoplasm of wild-type virus-infected cells. Although ICP0 is largely exported from nuclei late in infection, residual small granular structures reacting with anti-ICP0 antibody continue to dot the nuclear landscape. These structures are commonly seen in thin sections of infected cell nuclei, but this is the first indication that they contain ICP0. In an earlier study (28), the question was raised whether the export of ICP0 to the cytoplasm reflects the need to sequester viral proteins that have outlived their usefulness. While the small amount of ICP0 associated with marginated chromatin may express functions needed by the virus, the function of ICP0 within the dense aggregates is less clear.
(v) Last, the question arises as to how such functionally disparate proteins, such as gE and gI, which form the Fc heterodimer and the UL41 endoribonuclease, each independently effect the retention of ICP4 in the nucleus. We have no definitive data that bear on this question, but some aspects of the fundamental strategy of HSV-1 are relevant. Foremost, viral proteins perform several functions. Although the gE/gI complex and the UL41 protein have been linked to shuttling between the trans-Golgi network and the plasma membrane and to endoribonuclease activity, respectively, it is conceivable that they perform additional functions that at this time are less well defined. The second aspect of viral strategy is to sequester and redirect host proteins to perform novel functions. The gE/gI complex may, in the course of its shuttling activity, sequester the host protein that would be responsible for translocation of ICP4 to the cytoplasm.
Also, as noted in the introduction, there appears to be a connection between the UL41 protein and gE. Late in the course of the replicative cycle, the UL41 protein is neutralized by VP16 and VP22 (21, 23, 45, 49). There is ample evidence that VP22 forms complexes with a number of proteins that are ultimately packaged in virions. The assumption that this interaction occurs solely for the purpose of packaging and is not utilized by the virus for other objectives is probably incorrect. The more direct evidence of interaction may be deduced from the observation that in UL41-infected cells the accumulation of viral gene products occurs at a slower pace and in particular that the distribution of gE in UL41-infected cells lags behind that of wild-type virus-infected cells.
The notion that viral proteins are not merely directed by built-in signals or chaperoned to the nucleus but also specifically retained in the nucleus is intriguing. It overlays a novel level of regulation of viral gene functions.
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ACKNOWLEDGMENTS |
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We thank David C. Johnson of the Oregon Health Sciences University, Portland, OR, for the antibody to gE and Brunella Taddeo for helpful discussions.
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FOOTNOTES |
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Published ahead of print on 5 December 2007.
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